SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1’s ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.


Supplemental Figure 3│Ectopically expressed SOX11 co-localizes with endogenous SAMHD1 in inducible JVM-2
A. Top, representative pictures of proximity ligation assay performed on JVM-2 vector and JVM-2 iSOX11 using mouse monoclonal anti-SOX11 (MRQ-58) and rabbit polyclonal anti-SAMHD1 (Bethyl).Bottom, number of foci per cell and mean fluorescence intensity per cell.
Magnification 60x and pinhole is set at 1.2.This figure is related to Figure 1D-F (in the main manuscript).
B. Immunofluorescence confocal imaging of JVM-2 iSOX11 using mouse monoclonal anti-SAMHD1 (Abcam) and rabbit polyclonal anti-SOX11 (Abcam).To the right, Pearson correlation indicates the colocalization of the aligned staining channels for both proteins.This correlation was performed using ImageJ software.Magnification 100x and pinhole is set at 1.2.B. Cell cycle analysis of JVM-2 vector and JVM-2 iSOX11 at 24, 48 and 72 hours following treatment with 0.1 µM doxycycline.The number of cells in different cell cycle stages (G1, S, G2/M) are presented as percentage (%) of the selected cell population, with mean ± SEM of four independent biological repeats.An unpaired t-test was used to determine statistical differences between the amount of JVM-2 vector and JVM-2 iSOX11 cells in the different cell cycle stages; *P (Unpaired t test, two-tailed) <0.05.
C. Cell cycle analysis of JVM-2 vector and JVM-2 iSOX11 at 24, 48 and 72 hours in cells not treated with doxycycline.The number of cells in different cell cycle stages (G1, S, G2/M) are presented as percentage (%) of the selected cell population, with mean ± SEM of four independent biological repeats.An unpaired t-test was used to determine statistical differences between the amount of JVM-2 vector and JVM-2 iSOX11 cells in the different cell cycle stages.

D. Cell viability after 96 h of induction of SOX11 expression with 0.1 µM doxycycline.
Luminescence values of JVM-2 vector and JVM-2 iSOX11 cultured in different concentrations of doxycycline.The data are represented as mean ± SEM for four independent biological replicates.The P indicated on the chart are calculated by performing unpaired, two-tailed t-test with Welch correction.This figure is related to Figure 3D (in the main manuscript).

Supplemental Figure 6
Supplemental Figure 6│Sensitivity to ara-C in primary MCL samples with different

SOX11 expression and in JVM2 upon SOX11 induction.
A. Western blot shows SOX11, SAMHD1 and GAPDH in three primary MCL samples PS1-3 with different SOX11 expression levels (See Supplemental Table 1 for details).B, C. Dose-response curve for ara-C using PS1-3 primary MCL (two independent experiments).
The cells were treated with ara-C for 72h and cell viability was analyzed by CellTiter Glo assay.
The values on the y-axis represent the relative viability values which were calculated by normalizing absorbance value at each dose of ara-C for each condition to respective untreated controls (normalized as 100%), whereas treatment with high-dose doxorubicin was set as the 0%.(ab139418, Abcam, Netherlands), according to the manufacturer's protocol.Briefly, 2x10 5 cells were collected at specific time points, washed twice with PBS and fixed with 66% ethanol.Fixed cells were kept at 4°C until the last time point, and minimum 2 h before incubation with 1x Propidium Iodide plus RNase staining solution for 20 minutes at 37°C.Tubes containing the cells were then placed on ice until flow cytometry analysis using CytoFlex and the software FlowJo V10.A total of 1x10 4 cells were recorded for each sample, singlets and live cells were selected and the amount of PI staining per cell was assessed, displaying the number of cells in the different cell cycle phases.Statistical analysis was performed using GraphPad Prism 9.

Heterotopic JVM-2 animal model
On day 0, 64 mice were randomly assigned into two groups for cell injections: those receiving 10 6 JVM-2 iSOX11 cells (n = 32) and those receiving 10 6 JVM-2 vector cells (n = 32).Cells were injected subcutaneously on the flank in a 1:1 mixture of cells and Matrigel (Corning, 354234) in a total volume of 100 µL.On day 2, each group was randomly divided further into two groups: those receiving sterile water with 1 mg/mL Doxycycline (Sigma) (n = 16 for JVM-2 iSOX11 and n = 16 for JVM-2 vector ) and those receiving water without antibiotics (n = 16 for JVM-2 iSOX11 and n = 16 for JVM-2 vector ).On day 5, half of the mice from each group were randomly assigned to receive intraperitoneal injections of either ara-C (Cytarabine Accord, 100 mg/mL, Apoteket) 100 mg kg-1 (n = 8 for JVM-2 iSOX11 with Doxycycline water, n = 8 for JVM-2 iSOX11 with normal water, n = 8 for JVM-2 vector with Doxycycline water, n = 8 for JVM-2 vector with normal water) or PBS (n = 8 for JVM-2 iSOX11 with Doxycycline water, n = 8 for JVM-2 iSOX11 with normal water, n = 8 for JVM-2 vector with Doxycycline water, n = 8 for JVM-2 vector with normal water) consecutively for a total of 7 days.See Supplemental figure 8 for an overview of the groups.Tumour sizes were measured using calipers 3-5 times per week, and volume was estimated as tumour volume = 0.5 × tumour length × (tumour width). 1 General welfare was monitored daily, and animals were weighed at least two times per week.Mice were euthanized when tumour volume reached 1,500 mm 3 or length reached 20 mm.In a handful of cases, animals were euthanized due to tumour ulceration, which was determined to be severe after consulting with the veterinarian.Upon autopsy, tumours were excised and split into two equal parts for either flash-freezing with liquid nitrogen or fixation in 4% formaldehyde before further processing.Fixed tumours were paraffin-embedded, formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut using a microtome, and 4 µm-thick tissue slices were mounted on glass microscopy slides before deparaffination and rehydration using standard techniques.H&E staining was performed using standard procedures.For immunohistochemistry, antigen retrieval was following the protocol for UltraVision LP Detection System, HRP Polymer & DAB Plus Chromogen (Thermo Scientific).Slides were incubated overnight at 4°C with either 1:25 dilution of anti-SOX11 antibody (382M-14, Cell Marque) or 1:500 dilution of anti-SAMHD1 antibody (A303-691A, Bethyl) in 1% BSA in PBS.Slides were visualized using a Zeiss light microscope.
For each sample, 100 µL of Protein G Dynabeads (Invitrogen, Novex) were used.Briefly, beads were maintained at 4 °C, collected on a magnetic rack and re-suspended in fresh 0.5 % (w/v) BSA/PBS 3 times.Pre-blocked beads were incubated on a rotating platform at 4 °C O/N with 10 µg of anti-SOX11 (HPA000536, Sigma) or anti-IgG (Normal Rabbit IgG #2729, Cell Signaling Technology) in 250 µL 0.5 % (w/v) BSA/PBS.Antibody-saturated beads were washed 3 times in 0.5 % (w/v) BSA/PBS and re-suspended in 100 µL 0.5 % (w/v) BSA/PBS before added to the cell supernatant.The immunoprecipitation was carried out at 4 °C, O/N, on a rotating platform.

III. LC-ESI-LTQ-Orbitrap Analysis
Before analysis on the LTQ Orbitrap Velos (Thermo Fischer Scientific,), peptides were separated using an Agilent 1200 nano-LC system.Samples were trapped on a Zorbax 300SB-C18, and separated on a NTCC-360/100-5-153 (Nikkyo Technos Ltd) column using a gradient of A (3% ACN, 0.1% FA) and B (95% ACN, 0.1% FA), ranging from 3 % to 40% B in 180 min with a flow of 0.4 µL/min.The LTQ Orbitrap Velos was operated in a data dependent manner, selecting 5 precursors for sequential fragmentation by CID and HCD (Higher-energy Collisional Dissociation), and analyzed by the linear iontrap and orbitrap, respectively.The survey scan was performed in the Orbitrap at 30,000 resolution (profile mode) from 300-2000 m/z, using lock mass at m/z 445.120025, with a max injection time of 500 ms and AGC set to 1 x 10 6 ions.For generation of HCD fragmentation spectra, a max ion injection time of 500 ms and AGC of 5 x 10 4 were used before fragmentation with 325% normalised collision energy.
For FTMS (Fourier Transform Mass Spectrometry) MS2 spectra, normal mass range was used, centroiding the data at 7500 resolution.Peptides for CID were accumulated for a max ion injection time of 200 ms and AGC of 3 x 10 4 , fragmented with 35% collision energy, wideband activation on, activation q 0.25, activation time 10 ms before analysis at normal scan rate and mass range in the linear iontrap.Precursors were isolated with a width of 2 m/z and put on the exclusion list for 90 s.Single and unassigned charge states were rejected from precursor selection.

IV. Peptide and Protein Identification
All Orbitrap data was searched by SequestHT under the software platform Proteome Discoverer 1.4 (Thermo) against the Uniprot human database (140407) and filtered to a 1% false discovery rate (FDR).A precursor mass tolerance of 10 ppm, and product mass tolerances of 0.02 Da for HCD-FTMS and 0.36 Da for CID-ITMS (Ion trap mass spectrometry) were used.Further settings used were trypsin with 2 missed cleavages, iodoacetamide on cysteine, and oxidation of methionine as variable modification.
Figure 1D-F (in the main manuscript).

2 A
Figure3E(in the main manuscript).P (two-tailed) was calculated by Two-way ANOVA analysis to compare different conditions: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns P ≥ 0.05.Supplemental Figure 7Supplemental Figure7│SOX11induction reduces intracellular level of ara-CTP in JVM--B.Absolute intracellular levels of dNTPs and ara-CTP in pmol/million cells in JVM-2 vector or JVM-2 iSOX11 cells without (A) and with (B) ara-C (10 µM), after 24 h.Related to Figure3G(in the main manuscript).C.Intracellular ara-CTP levels normalized to levels of canonical dATP (left), dGTP (middle) or dCTP (right), determined using HPLC-MS/MS.Both JVM-2 vector and JVM-2 iSOX11 were treated with 10 µM of ara-C for 24 h.Circles and error bars correspond to individual values, mean ± SEM of at three independent experiments.Analyses were performed using unpaired two-tailed t-tests.*P < 0.05.This figure is related to Figure3G(in the main manuscript).Supplemental Figure 8Supplemental Figure8│SOX11 induction in the presence of ara-C significantly reduced tumour sizes in mice injected with JVM-2 cells A. Illustration of stratification of xenotransplated mice with JVM-2 vector or JVM-2 iSOX11 and plan for ara-C and doxy treatment.B. Average tumor volumes during and after ara-C treatment in mice injected with iSox11 cells treated with ara-C and doxycycline (black) and mice treated with ara-C alone (red).Lines represent average volumes for the groups.Statistical analysis was done using unpaired twotailed t-test of the means.t = 6.057, df = 11, ****P < 0.0001.C. Average tumor volumes during and after ara-C treatment in mice injected with iSox11 cells treated with ara-C and doxycycline (black) versus mice injected with vector cells treated with ara-C and doxycycline (blue).Lines represent average volumes for the groups.Statistical analysis was done using unpaired two-tailed t-test of the means.t = 3.309, df = 8, *P < 0.05.Supplemental Figure 9 Supplemental Figure 9│HU synergizes with ara-C in SOX11-negative JVM-2 A, B. Synergy analyses for the addition of HU to ara-C in JVM-2 vector (A) and JVM-2 iSOX11 (B) cells using zero interaction potency, with heat maps showing areas of most synergy in red.Areas of highest synergy highlighted with concentrations of HU and ara-C on the x-and y-axis, respectively.C-F.Semiquantitative analysis of Δ increase in the level of the indicated markers of apoptosis and DNA damage upon single treatment of HU or ara-C or their combination versus the respective untreated controls (related to Figure 4E in the main manuscript).The data are represented as mean ± SEM for three independent biological replicates.